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Caffeine in Citrus flowers

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The allocation of purine alkaloids within citrus flowers was studied and found to be linked to anthesis, with 99% of the total flower caffeine confined to the androecium. The main alkaloid is caffeine accompanied by considerable (up to 30% of caffeine) concentrations of theophylline. In the anther, these purine alkaloids reach altogether a concentration of 0.9% dry wt which is close to the caffeine content of the Arabica coffee bean. The pollen alkaloid concentration is in the same range. Much lower but still marked concentrations were found in the nectar. A considerable breakdown of alkaloids during honey production is assumed. The biological significance of this particular secondary compound allocation as well as possible effects on the key pollinator, the honey-bee, are discussed.
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Caeine in Citrus ¯owers
Josef A. Kretschmar, Thomas W. Baumann*
Institute of Plant Biology, University of Zu
Èrich, Zollikerstr. 107, 8008 Zu
Èrich, Switzerland
Received 11 August 1998; received in revised form 4 January 1999; accepted 21 January 1999
Abstract
The allocation of purine alkaloids within citrus ¯owers was studied and found to be linked to anthesis, with 99% of the total
¯ower caeine con®ned to the androecium. The main alkaloid is caeine accompanied by considerable (up to 30% of caeine)
concentrations of theophylline. In the anther, these purine alkaloids reach altogether a concentration of 0.9% dry wt which is
close to the caeine content of the Arabica coee bean. The pollen alkaloid concentration is in the same range. Much lower but
still marked concentrations were found in the nectar. A considerable breakdown of alkaloids during honey production is
assumed. The biological signi®cance of this particular secondary compound allocation as well as possible eects on the key
pollinator, the honey-bee, are discussed. #1999 Elsevier Science Ltd. All rights reserved.
Keywords: Citrus; Rutaceae; Caeine; Theophylline; Purine alkaloids; Flower development; Nectar; Pollen; Apis mellifera; Honey-bee
1. Introduction
The compilations often found in literature (e.g.
Willaman & Schubert, 1961), regarding the occurrence
of caeine may give the impression that this purine al-
kaloid is shared by a large number of genera.
However, if we rely exclusively on data re-examined by
advanced analytical techniques, we arrive at the con-
clusion that during evolution, 'invention` of caeine,
i.e. the purine alkaloid pathway, was a relatively rare
event meaning that out of ca. 10,000 angiosperm gen-
era only seven developed, to our present knowledge,
this phytochemical feature, namely Coea, Camellia,
Theobroma, Herrania, Cola, Ilex, and Paullinia.
Therefore, a report by Stewart in 1985 (Stewart, 1985)
was most exciting because it claimed the presence of
caeine, even though in the very low range of 6 and
50 ppm (31 and 258 nmol g
ÿ1
fr. wt) in leaves and
¯owers, respectively, of several Citrus species. His ®nd-
ings were con®rmed by an Italian group (Trova,
Cossa, & Gandolfo, 1994) which detected caeine in
dried citrus ¯owers (237 to 856 nmol g
ÿ1
dry wt) com-
mercially available for preparing a tea. Moreover, the
authors found caeine for the ®rst time also in honeys
originating from the activities of honey-bees (Apis mel-
lifera ) visiting either frequently (uni¯oral honey) or
sporadically ('mille®ori` honey) the ¯owers in orange
plantations. Caeine in these honeys ranged from 2.6
to 52 nmol g
ÿ1
. Later, the analyses were extended to
various other citrus honeys (De®lippi, Piancone, &
Tibaldi, 1995; Vacca & Fenu, 1996) and possible
source-¯owers (Vacca, Agabbio, & Fenu, 1997) with
the aim to establish a measure of quality control.
However, a correlation could not yet be established. In
all these studies, neither single ¯ower organs nor nec-
tar and pollen were examined.
In a preliminary investigation on coee (Kretschmar
& Baumann, 1998) we recognised that ¯ower caeine
was relatively abundant (ca. 3200 nmol g
ÿ1
fr. wt) and
preferentially, even though not very markedly, allo-
cated in the androecium together with other purine al-
kaloids. Therefore, we analysed the within-the-¯ower
distribution of these alkaloids in citruses (including
Poncirus ) considered ideal to demonstrate organ-
speci®c allocation because of the low average content.
Indeed, among the ¯ower organs analysed the androe-
cium had by far the highest concentration of alkaloids,
Phytochemistry 52 (1999) 19±23
0031-9422/99/$ - see front matter #1999 Elsevier Science Ltd. All rights reserved.
PII: S0 0 3 1 - 9 4 2 2 ( 9 9 ) 0 0 1 1 9 - 3
* Corresponding author. Fax: +41-1-634-8204.
E-mail address: twb@botinst.unizh.ch (T.W. Baumann)
with a main allocation in the anther and pollen at a
very high level. Moderate, but still marked purine al-
kaloid concentrations were found in the nectar. Since
caeine is known to be insecticidal, the results were
discussed also in the context of intoxication of honey-
bees.
2. Results and discussion
In a ®rst approach the purine alkaloid content of
entire ¯owers of Citrus paradisi and C. maxima was
determined. Caeine was the main alkaloid (21 and 77
nmol g
ÿ1
fr. wt, respectively) accompanied by theo-
phylline (4 and 15 nmol g
ÿ1
fr. wt, respectively), while
theobromine and paraxanthine occurred in traces only.
Similarly, commercially available orange ¯ower tea (C.
sinensis ) contained caeine (182 nmol g
ÿ1
dry wt) and
theophylline (46 nmol g
ÿ1
dry. wt). These caeine
values are in the range as found before (Stewart, 1985;
Trova et al., 1994), but until now the presence of theo-
phylline and other dimethylxanthines in Citrus has not
been reported. Theophylline is a trace compound in
the `classical' caeine plants consumed by the human.
In some of the citrus ¯ower tissues it showed consider-
able accumulation (Table 1).
The chemical analysis of ¯ower development (C.
limon ) revealed, that the small, round-shaped ¯ower
bud was virtually alkaloid-free, with only a trace of
theophylline, whereas the elongated bud shortly before
anthesis contained well-measurable concentrations of
both caeine and theophylline, accompanied by little
theobromine and paraxanthine. During anthesis the
caeine content increased by a factor of almost 2
(Table 1). Similarly (not shown), two stages (145 and
216 mg fr. wt) of ¯ower buds of the closely related
Poncirus trifoliata were purine alkaloid-free, whereas
the freshly opened ¯ower (375 mg fr. wt) had an over-
all caeine concentration of 109 nmol g
ÿ1
fr. wt. A
similar increase was observed during anthesis of C.
paradisi. Therefore we may conclude that anthesis in
citruses is coordinated with a rapid allocation of caf-
feine.
Then, the ¯owers of C. paradisi,C. maxima,,C.
limon and P. trifoliata were separated into petals, pis-
tils and stamens and analysed (the tiny green sepals
and the ¯ower base were found in preliminary exper-
iments to be virtually alkaloid-free). Both, petals and
pistils contained very small concentrations (mostly in
the range of 2 to 10 nmol g
ÿ1
fr. wt, respectively) of
caeine, theobromine, or theophylline (not shown).
However, the stamens (Table 1) contained the highest
concentrations of caeine and theophylline and were
the exclusive site of ¯ower paraxanthine which was
hardly detectable in the related entire ¯owers because
of dilution. The separate analysis of ®lament and
anther revealed maximum alkaloid concentrations in
the latter exceeding altogether the concentration of
Table 1
Purine alkaloid content (nmol g
ÿ1
fr. wt) in ¯owers, nectars and honeys of Citrus spp. n= 3±10 (¯owering units); n.d.=not detectable
Caeine Theobromine Theophylline Paraxanthine
Flower development (C. limon)
Small bud (148 mg; n= 5) n.d. n.d. 621
a
n.d.
Large bud (840 mg; n= 3) 16622
a
252251
Full anthesis (938 mg; n= 3) 318231 27211
Stamens
C. paradisi 3'233242 28 1732222
C. maxima 1'110213 13 305 245
P. trifoliata 807211 5 56215
C. limon 1'415218 67 115228
Filament
C. paradisi 1'917229 18 1392215
C. maxima 850211 8 12 214
Anther
C. paradisi 8'5512169 26 1'491229 32
C. maxima 7'9002119 18 2'753 241 20
Pollen
C. medica 6'8572321 n.d. 1'921290 n.d.
Nectar (nmol ml
ÿ1
)
C. paradisi 487215 22 552212
C. maxima 912510 3213
C. limon 6022 n.d. n.d. n.d.
Honey (nmol g
ÿ1
)
Sicilia 31216 3213
California 221 1 n.d. n.d.
a
Mean value (2the estimated experimental error, see Section 3).
J.A. Kretschmar, T.W. Baumann / Phytochemistry 52 (1999) 19±2320
10,000 nmol (=10 mmol) per g fr. wt. If related to dry
wt, it results a value of ca. 0.7±0.8% caeine and
0.9% total purine alkaloid in the anther. Hence, the
purine alkaloid concentration in the anther is close to
that in the Arabica coee bean (1.2%, almost exclu-
sively caeine)!
Finally, pollen of (due to the absence of blooming
of the other species) Citrus medica (citron) was ana-
lysed. This single analysis revealed a high purine alka-
loid concentration (altogether almost 8800 nmol g
ÿ1)
in the microspores (Table 1), which is in the range of
that found in the anther of the closely related species.
We cannot yet decide whether the anther alkaloid
amount is completely con®ned to the pollen, or
whether the anther wall contains alkaloid at concen-
trations similar to pollen. Also, we have not yet stu-
died the localisation of purine alkaloids within the
pollen grain. At the present we can only speculate
about the signi®cance of this conspicuous allocation.
Besides protection against (unknown) pollen predators,
purine alkaloids are well-studied defence compounds
(reviewed in Harborne (1993)), the cytokinin-like eect
of caeine (Vito
Âria & Mazzafera, 1997) may play a
role during pollination and seed set in citrus
(Hernandez Minana & Primo Millo, 1990).
Blossom honey essentially consists of nectar concen-
trated by the activities of specialised bees in the hive.
The pollen present in the honey is quantitatively negli-
gible but a valuable nectar 'contaminant` which is of
help to trace the source ¯owers. It has been reported
that on average 64% of the pollen found in uni¯oral
citrus honeys is citrus pollen (White & Bryant, 1996)
meaning that roughly two third of the honey originate
from citrus nectar. Since the latter was shown to con-
tain caeine in the range of ca. 60 to 490 nmol ml
ÿ1
(Table 1) and undergoes a concentration process by a
factor of ca. 2 during honey production, one should
expect a much higher caeine concentration in citrus
honey than found in the present (2 and 31 nmol g
ÿ1
;
Table 1) or in earlier (2.5±50 nmol g
ÿ1
(Trova et al.,
1994)) studies. It can be calculated that about 95% of
the nectar caeine is removed or degraded by a still
unknown mechanism. However, even though the nec-
tar was sampled with caution we cannot rule out con-
tamination by pollen.
In order to obtain an estimate of chemical defence
allocation, the number and weight of the individual
¯ower organs were determined and the alkaloid distri-
bution calculated as exempli®ed for C. lemon in Table
2. In summary, one citrus ¯ower contains about 260
nmol (ca. 50 mg) caeine and 300 nmol total purine al-
kaloid, 99 and 96.6%, respectively, allocated to the
androecium! The amount of alkaloids found in the
nectar (ca. 20 ml) at the moment of ¯ower dissection is
negligible in the case of C. lemon (ca. 1 nmol; caeine
only), but was distinctly higher in C. paradisi (ca. 10
nmol) and may be considerable if extrapolated to the
entire period of ¯owering. However, nectar secretion
was not studied in detail. Diurnal ¯uctuations are to
be expected and may account for some of the dier-
ences in pattern and concentrations of nectar purine
alkaloids listed in Table 1.
Finally, we should mention that orange ¯ower tea
(Aurantii ¯os) is pharmaceutically recommended for
treatment of sleeplessness [O
ÈAB, Ph. Helv. VI]. The
amount of caeine ingested by consumption of such a
calming tea is below 100 mg, a dose present in homeo-
pathic coee preparations used against insomnia
(HAB, (Baumann & Seitz, 1992)).
2.1. Caeine and honey-bees
High concentrations of secondary compounds have
been detected in microspores of both wind- (e.g.
Meurer, Wray, Wiermann, & Strack, 1988) and insect-
(reviewed in Detzel & Wink, 1993) pollinated ¯owers.
In the latter case the question of intoxication of the
pollinators arises. Detzel and Wink (Detzel & Wink,
1993) tested a large number of such allelochemicals on
the feeding behaviour of honey-bees. Caeine was
found to act as a deterrent and its toxicity was com-
paratively low under no-choice conditions (LD
50
at 0.2
%). In an earlier study (Ishay & Paniry, 1979), honey-
bees, oered free choice of either the sugar solution
alone or the sugar solution with the caeine, similarly
preferred the sugar solution. The concentration of the
Table 2
Purine alkaloid allocation within the ¯ower of Citrus lemon. The ¯ower base is virtually alkaloid-free
Organ Caeine aPurine alkaloids
fr. wt (mg) nmol g
ÿ1
fr. wt amount (nmol) % of total nmolg
ÿ1
fr. wt Amount (nmol) % of total
Petals (4±5) 509 3.9 2.0 0.8 15.3 7.8 2.5
Stamens (31±32) 183 1415.0 258.9 99.1 1604.5 293.6 96.5
Pistil (1) 103 2.5 0.3 0.1 29.5 3.0 1.0
Flower base (1) 119 n.d. 0 ± ± ± ±
Total 914 285.8
a
261.2 100 333.0
a
304.4 100
a
Overall concentration of the entire ¯ower. In parenthesis: number of organs per one ¯ower. For nectar see text.
J.A. Kretschmar, T.W. Baumann / Phytochemistry 52 (1999) 19±23 21
caeine solution (ca. 250 mM) happened to be in the
range of citrus nectars (Table 1). It was readily
accepted under no-choice conditions. After ®ve days, a
300±500 % boost in oviposition by the (young) queen,
an enhanced activity of the bees outside the hive, and
an improved defence by bees against hornets at the
hive entrance was observed. In contrast, hornets also
fed with caeine ceased foraging in the ®eld and also
failed to clear the dead (poisoned by caeine) larvae
out of the nest. On the cellular level, caeine was
shown to in¯uence the cytosolic Ca
2+
concentration in
various organisms including honey-bees, where the caf-
feine-sensitive Ca
2+
release from the endoplasmic reti-
culum in photoreceptors has been studied (Walz,
Baumann, & Ciriacy-Wantrup, 1994).
Caeine and related substances are known not only
to exert insecticidal activity but also to synergize the
eects of pesticides (Nathanson, 1984). On the one
side it appears that the toxicity of caeine in the
honey-bee larvae, which to our knowledge do not suf-
fer from the caeine-rich pollen in citrus orchards, is
relatively low. On the other side, however, one should
in future evaluate bee toxicity of pesticides also in the
presence of purine alkaloids, in order to account for
the above-mentioned synergistic eect which may
occur not only in citrus but also in coee and tea plan-
tations.
Finally, we should mention that recently citrus pol-
len was found to be toxic to the predatory mite
Euseius mesembrinus (Yue, Childers, & Fouly, 1994)
which is a facultative pollen feeder widely distributed
in citrus plantations. If toxicity is due to the presence
of purine alkaloids, one may suggest that mites, in
contrast to honey-bees, are very susceptible to purine
alkaloids, a situation which could be advantageous in
the chemical control of the ectoparasitic mite Varroa
jacobsoni associated with the honey-bee.
3. Experimental
3.1. Plant material
Citrus plants (C. paradisi Macf., grapefruit; C. max-
ima (Burm.) Merr., shaddock; C. limon (L.) Burm.f.,
lemon, and C. medica L., citron) were grown in the
greenhouse. The trifoliate orange plant (P. trifoliata
Raf.) was kept outdoors in the institute garden. Tea of
orange ¯owers (C. sinensis (L.) Pers. was bought in a
local store (Coop, Switzerland) as well as orange
¯ower honeys of Sicilian (Globus, Switzerland) and
Californian (Biorex AG, Ebnat-Kappel, Switzerland)
origin. Nectar was sampled using a glass (hematokrit)
capillary.
3.2. Purine alkaloid extraction
Fresh entire ¯owers (n= 3 to 10) or the related
¯ower parts were pooled and extracted in 0.1 N HCl
(1 ml per 50 to 150 mg fr. wt) at 508for 30 min by
sonication. One ml of the extract was applied onto a
Kieselgur column (Extrelut1, Merck). Essential oil was
removed by 12 ml hexane and thereafter purine alka-
loids were eluted with 12 ml CH
2
Cl
2
. The eluate was
dried under a stream of N
2
and the residue dissolved,
for HPLC, in 1 ml H
2
O. Orange ¯ower tea was
extracted likewise (1 ml 0.1 N HCl per 250 mg, with-
out further drying the ¯owers) as well as honey (1 g),
which was diluted with 0.1 N HCl (2 ml) prior to ap-
plication onto the Kieselgur column. Nectar was
mixed (1:1) with 8% MeOH and directly injected.
Pollen was collected and processed as follows: stamens
were harvested, dried at room temp. and transferred
into a pre-weighed Eppendorf tube, which then was
vortexed at high speed to spin o the pollen. The sta-
mens were removed and the weight of the pollen was
determined by weighing the tube again (Mettler AE
240). The pollen (2.84 mg) was suspended in 300 ml 0.1
N HCl. After 3 h at room temp. a 30-min-sonication
at 408followed. Thereafter, the suspension was ®ltered
through a membrane ®lter and directly injected into
HPLC. No attempt was made to determine the dry
weight of the pollen.
3.3. HPLC separation of purine alkaloids
HPLC separation of purine alkaloids was carried
out on a Nucleosil-100-5 C18 HD column; precolumn
48 mm; ChromCart, Macherey-Nagel). Parameters
were controlled by a Hewlett-Packard liquid chromato-
graph equipped with a diode array detector set at 272
nm. Chromatography was carried out using the follow-
ing gradient: 0±4 min with 0±7.5% MeOH and 0±
2.5% AcN, 4±20 min with 7.5% MeOH and 2.5%
AcN. The R
t
's (min) were 7.1, 9.1, 9.6 and 16.0 for
theobromine, paraxanthine, theophylline, and caeine,
respectively. The ¯ow rate was 1.1 ml min
1
and injec-
tion vol 150 ml. Peak identi®cation was achieved by
comparing UV spectrum (library established under
separating conditions) and retention time of authentic
standards.
3.4. Calculation of the mean value and the `systematic
experimental error'
Since in this study we did not aim at showing the
variability of purine alkaloids and their concentrations
among ¯owers from one or several plants, the ¯owers
were randomly collected and pooled (with ngener-
ally=10, in a few cases of shortage lower but never
less than 3). The obtained mean value has an exper-
J.A. Kretschmar, T.W. Baumann / Phytochemistry 52 (1999) 19±2322
imental error which was calculated by considering the
accuracy of the fresh weight determination (21 mg)
and the quantitation by HPLC (21%).
Acknowledgements
We thank the beekeepers Kurt Honegger, Baden
and Pierre Pittier, Dietlikon, for answering awkward
questions of botanists and Stefan Bogdanov, Federal
Dairy Research Institute, Bee Department, Bern
Liebefeld, Switzerland, for his expertise in bee-related
topics as well as for critically reading the manuscript.
This work was ®nancially assisted by the Swiss
National Science Foundation, Grant No. 31-50521.97
and by the `Jubila
Èumsspende fu
Èr die Universita
Èt
Zu
Èrich'.
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J.A. Kretschmar, T.W. Baumann / Phytochemistry 52 (1999) 19±23 23
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... Paraxanthine (1,7-dimethylxanthine, PXN) accounts for 70-72% of CA ingested, and 85% of the methylxanthine metabolic by-products [1], with smaller percentages of CA being metabolized to theobromine (3,7-dimethylxanthine, TB) and theophylline (1,3dimethylxanthine, TP). PXN is a natural dietary component that can be found in Theobroma cacao fruits [2], Coffea arabica [3], Sinomenium actum [4], and the stamens of citrus flowers [5]. The pharmacokinetics of TB and TP are similar and significantly different from CA and PXN, with PXN having the shortest half-life and greatest plasma clearance [6]. ...
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This study examined the effects of acute paraxanthine (PXN) ingestion on markers of cognition, executive function, and psychomotor vigilance. In a randomized, double blind, placebo-controlled, crossover, and counterbalanced manner, 13 healthy male and female participants were randomly assigned to consume a placebo (PLA) or 200 mg of PXN (ENFINITY™, Ingenious Ingredients, L.P.). Participants completed stimulant sensitivity and side effect questionnaires and then performed the Berg Wisconsin Card Sorting Test (BCST), the Go/No-Go test (GNG), the Sternberg task test (STT), and the psychomotor vigilance task test (PVTT). Participants then ingested one capsule of PLA or PXN treatment. Participants completed side effect and cognitive function tests after 1, 2, 3, 4, 5, and 6 h after ingestion of the supplement. After 7 days, participants repeated the experiment while consuming the alternative treatment. Data were analyzed by general linear model (GLM) univariate analyses with repeated measures using body mass as a covariate, and by assessing mean and percent changes from baseline with 95% confidence intervals (CIs) expressed as means (LL, UL). PXN decreased BCST errors (PXN −4.7 [−0.2, −9.20], p = 0.04; PXN −17.5% [−36.1, 1.0], p = 0.06) and perseverative errors (PXN −2.2 [−4.2, −0.2], p = 0.03; PXN −32.8% [−64.4, 1.2], p = 0.04) at hour 6. GNG analysis revealed some evidence that PXN ingestion better maintained mean accuracy over time and Condition R Round 2 response time (e.g., PXN −25.1 [−52.2, 1.9] ms, p = 0.07 faster than PLA at 1 h), suggesting better sustained attention. PXN ingestion improved STT two-letter length absent and present reaction times over time as well as improving six-letter length absent reaction time after 2 h (PXN −86.5 ms [−165, −7.2], p = 0.03; PXN −9.0% [−18.1, 0.2], p = 0.05), suggesting that PXN enhanced the ability to store and retrieve random information of increasing complexity from short-term memory. A moderate treatment x time effect size (ηp2 = 0.08) was observed in PVTT, where PXN sustained vigilance during Trial 2 after 2 h (PXN 840 ms [103, 1576], p = 0.03) and 4 h (PXN 1466 ms [579, 2353], p = 0.002) compared to PL. As testing progressed, the response time improved during the 20 trials and over the course of the 6 h experiment in the PXN treatment, whereas it significantly increased in the PL group. The results suggest that acute PXN ingestion (200 mg) may affect some measures of short-term memory, reasoning, and response time to cognitive challenges and help sustain attention.
... This showed that carbendazim treatment can increased the metabolism of FDP, thereby protecting the brains of bees. The 3-methylxanthine is the product of caffeine metabolism conversion (Ashihara and Crozier, 1999) and occurs due to caffeine in the pollen of plant (Kretschmar and Baumann, 1999). Caffeine had no effect on acquisition, but increased long term memory retention in an appetitive visual learning task in the honey bee (Si et al., 2005). ...
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Pesticides are one of the main causes of colony losses globally, posing a huge threat to the beekeeping industry. The fungicide carbendazim is commonly used on many crops worldwide, but the effects of fungicides on honey bees have received less attention than those of insecticides. Previous studies have shown that sublethal doses of carbendazim hinder growth and development and may destabilize and impede the development of honey bee colonies. The metabolome closely reflects brain activity at the functional level, allowing the effects of compounds such as fungicides to be investigated. Here, we established a model of carbendazim-treated honey bees, Apis mellifera, and used metabolomic approaches to better understand the effect of carbendazim on bee metabolic profiles. The results showed that 112 metabolites were significantly affected in carbendazim-treated bees compared to the control. Metabolites associated with energy and amino acid metabolism showed high abundance and were enriched for a wide range of pathways. In addition, the down-regulation of Aflatoxin B1exo-8,9-epoxide-GSH,(5B,12A) and glycerol diphosphate showed that carbenazim may affect the detoxification and immune system of honey bees. These results provide new insights into the interaction between fungicides and honey bees.
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Citrus maxima (Burm). Merr. (family Rutaceae), commonly known as Pomelo, is an ethnomedicinally, pharmacologically, and phytochemically valued species. Various ethnomedicinal reports have revealed the use of C. maxima for cough, fever, asthma, diarrhea, ulcer, and diabetes and as a sedative. Numerous phytochemicals have been reported from C. maxima such as polyphenols, terpenoids, sterols, carotenoids, vitamins, and amino acids. The plant possesses significant bioactivities like antioxidant, antimicrobial, anti-inflammatory, analgesic, anticancer, antidiabetic, anti-Alzheimer’s disease, insecticidal, anxiolytic, hepatoprotective, antimalarial, and antiobesity. Extensive research is necessary to explore the detailed mechanism of action of extracts and compounds to design effective medicines, herbal products, and functional foods.
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A unique aspect of metabolic detoxification in insects compared to other animals is the presence of xenobiotic phosphorylation, about which little is currently understood. Our previous work raised the hypothesis that members of the taxonomically restricted ecdysteroid kinase-like (EcKL) gene family encode the enzymes responsible for xenobiotic phosphorylation in the model insect Drosophila melanogaster (Diptera: Ephydroidea)—however, candidate detoxification genes identified in the EcKL family have yet to be functionally validated. Here, we test the hypothesis that EcKL genes in the rapidly evolving Dro5 clade are involved in the detoxification of plant and fungal toxins in D. melanogaster. The mining and reanalysis of existing data indicated multiple Dro5 genes are transcriptionally induced by the plant alkaloid caffeine and that adult caffeine susceptibility is associated with a novel naturally occurring indel in CG31370 (Dro5-8) in the Drosophila Genetic Reference Panel (DGRP). CRISPR-Cas9 mutagenesis of five Dro5 EcKLs substantially decreased developmental tolerance of caffeine, while individual overexpression of two of these genes—CG31300 (Dro5-1) and CG13659 (Dro5-7)—in detoxification-related tissues increased developmental tolerance. In addition, we found Dro5 loss-of-function animals also have decreased developmental tolerance of the fungal secondary metabolite kojic acid. Taken together, this work provides the first compelling functional evidence that EcKLs encode detoxification enzymes and suggests that EcKLs in the Dro5 clade are involved in the metabolism of multiple ecologically relevant toxins in D. melanogaster. We also propose a biochemical hypothesis for EcKL involvement in caffeine detoxification and highlight the many unknown aspects of caffeine metabolism in D. melanogaster and other insects.
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Paraxanthine or 1,7-dimethylxanthine is a natural dietary component and the main metabolite of caffeine in humans. A battery of toxicological studies was conducted in accordance with international guidelines to investigate mutagenicity, genotoxicity and acute and repeated-dose oral toxicity in rats of synthetic paraxanthine (ENFINITY™, Ingenious Ingredients, L.P., >99% purity). There was no evidence of mutagenicity in a bacterial reverse mutation as well as in an in vitro mammalian chromosomal aberration test. There was no evidence of genotoxicity in an in vivo mammalian erythrocyte micronucleus test as well as in an in vitro mammalian cell gene mutation test. An acute oral toxicity test resulted in a LD50 value of 1601 mg/kg bw/d. Paraxanthine did not cause mortality or toxic effects in a subacute 28-day repeated-dose oral toxicity study at daily doses of 75, 150, or 300 mg/kg bw/d (each group n = 10 per sex), administered by gavage. Paraxanthine also did not cause mortality or toxic effects in a subchronic 90-day repeated-dose oral toxicity study at daily doses of 75, 150, or 300 mg/kg bw/d (each group n = 10 per sex), administered by gavage. The no observed adverse effect level (NOAEL) determined from the 90-day study was greater than or equal to 300 mg/kg bw/d, the highest dose tested, for both male and female Wistar rats.
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Caffeine is a widely occurring plant defense chemical¹,² that occurs in the nectar of some plants, e.g., Coffea or Citrus spp., where it may influence pollinator behavior to enhance pollination.³,⁴ Honey bees fed caffeine form longer lasting olfactory memory associations,⁵ which could give plants with caffeinated nectar an adaptive advantage by inducing more visits to flowers. Caffeinated free-flying bees show enhanced learning performance⁶ and are more likely to revisit a caffeinated target feeder or artificial flower,7, 8, 9 although it is not clear whether improved memory of the target cues or the perception of caffeine as a reward is the cause. Here, we show that inexperienced bumble bees (Bombus terrestris) locate new food sources emitting a learned floral odor more consistently if they have been fed caffeine. In laboratory arena tests, we fed bees a caffeinated food alongside a floral odor blend (priming) and then used robotic experimental flowers¹⁰ to disentangle the effects of caffeine improving memory for learned food-associated cues versus caffeine as a reward. Inexperienced bees primed with caffeine made more initial visits to target robotic flowers emitting the target odor compared to control bees or those primed with odor alone. Caffeine-primed bees tended to improve their floral handling time faster. Although the effects of caffeine were short lived, we show that food-locating behaviors in free-flying bumble bees can be enhanced by caffeine provided in the nest. Consequently, there is potential to redesign commercial colonies to enhance bees’ forage focus or even bias bees to forage on a specific crop.
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Citrus species are one of the major cultivated crops throughout the globe and have broad economic and pharmaceutical importance. Citrus-based products are used in various food and medicinal industries due to their strong fragrance and therapeutic properties. They are rich in micro as well as macronutrients. Analysis of their compounds has led to the identification of biologically active components which include flavonoids and alkaloids that contribute to pharmaceutical prominence. In this review, a systematic analysis of the research has been carried out on the utility of citrus and metabolites, during the past decade. Prominent actions like anti-oxidant, anti-inflammatory, anti-coagulant, anti-cancer and anti-venom properties of citrus are also compiled to enable further in-depth studies.
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Cytokinin amounts were determined in the ovaries of flowers and fruitlets on leafy and leafless inflorescences of seeded and seedless cultivars of Citrus sinensis, C. reshni and C. clementine. Flowers were sampled over a 65 day period from before anthesis to after fruit set, during the period from fructification to fruitlet expansion. Cytokinin activity appeared in both organic and aqueous phases obtained in the extraction process. Generally, the maximum activity in the organic phase occurred at anthesis, except in ‘Navelate’ orange, and decreased sharply following petal fall, whereas the aqueous phase had more cytokinins later, when the fruitlets had reached 1-2 cm diameter. The effect of pollination on cytokinin-like compounds was studied in seeded cultivars. The pollinated ovaries showed a peak in cytokinin activity at anthesis, in contrast to unpollinated ovaries. Partition chromatography of the organic phase on Sephadex LH-20 indicated five zones of cytokinin activity, four of which had the same elution volumes as ribosyl zeatin, zeatin, isopentenyl adenosine and isopentenyl adenine.
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Twenty-nine Sardinian honeys of different botanical origin (Orange, Citrus spp., Asphodel, Cardus spp., Strawberry-tree, Eucalyptus, Multiflowers, Chestnut-tree, Trifolium spp., Edisarum spp., and Mediterranean native flora) currently available on the market, were analyzed in order to determine the caffeine content. The results have shown an average concentration of caffeine of 1.79 and 4.93 mg/ kg in Citrus and Orange honeys respectively. On the contrary, the component was not found in the other honeys. According to previous results obtained by other Authors, these data confirm the peculiarity of Orange and Citrus honeys; therefore their botanical origin can be identified using the caffeine presence and concentration.
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Twenty-five samples of different species of citrus flowers (orange, lemon, grapefruit, tangerine, mandarin, mandarin-like and two hybrids) were tested for caffeine content. Although caffeine was found in all samples, the high variability in content within the samples did not allow a precise distinction to be made between the species. Caffeine concentration ranged from 4.61 mg/kg dry matter in a grapefruit sample (cv. "Star Ruby") to 30.92 mg/kg in a tangerine sample (cv. "Clementine di Nules"). The presence of the alkaloid confirms that caffeine found in orange and citrus honeys probably comes from flowers and is transferred to the honey by bees. However, the results of this study do not permit the origin of orange and citrus honeys to be determined from their caffeine content.
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Light stimulation of invertebrate microvillar photoreceptors causes a large rapid elevation in Cai, shown previously to modulate the adaptational state of the cells. Cai rises, at least in part, as a result of Ins(1,4,5)P3-induced Ca2+ release from the submicrovillar endoplasmic reticulum (ER). Here, we provide evidence for Ca(2+)-induced Ca2+ release (CICR) in an insect photoreceptor. In situ microphotometric measurements of Ca2+ fluxes across the ER membrane in permeabilized slices of drone bee retina show that (a) caffeine induces Ca2+ release from the ER; (b) caffeine and Ins(1,4,5)P3 open distinct Ca2+ release pathways because only caffeine-induced Ca2+ release is ryanodine sensitive and heparin insensitive, and because caffeine and Ins(1,4,5)P3 have additive effects on the rate of Ca2+ release; (c) Ca2+ itself stimulates release of Ca2+ via a ryanodine-sensitive pathway; and (d) cADPR is ineffective in releasing Ca2+. Microfluorometric intracellular Ca2+ measurements with fluo-3 indicate that caffeine induces a persistent elevation in Cai. Electrophysiological recordings demonstrate that caffeine mimics all aspects of Ca(2+)-mediated facilitation and adaptation in drone photoreceptors. We conclude that the ER in drone photoreceptors contains, in addition to the Ins(1,4,5)P3-sensitive release pathway, a CICR pathway that meets key pharmacological criteria for a ryanodine receptor. Coexpression of both release mechanisms could be required for the production of rapid light-induced Ca2+ elevations, because Ca2+ amplifies its own release through both pathways by a positive feedback. CICR may also mediate the spatial spread of Ca2+ release from the submicrovillar ER toward more remote ER subregions, thereby activating Ca(2+)-sensitive cell processes that are not directly involved in phototransduction.
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Hydroxycinnamic acid—spermidine amides have been isolated from pollen and identified from their 1H NMR and mass spectral data: N1,N5-di-(E)-p-coumaroylspermidine from Pterocarya fraxinifolia (Lam.) Spach., N5,N10-di-(E)-feruloylspermidine from Betula verrucosa Ehrh. a mixture of two di-(E)-p-coumaroylspermidines from Alnus glutinosa (L.) Gaertn.
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Effects on development, survivorship and reproduction of Euseius mesembrinus (Dean) were studied in the laboratory using nine different plant pollens: ice plant, Malephora crocea (Jacquin); live oak, Quercus virginiana Miller; castor bean, Ricinus communis L.; cattail, Typha latifolia L.; Spanish needle, Bidens pilosa L.; grapefruit, Citrus paradisi (L.) Osbeck; ‘Sunburst’ tangerine, C. reticulata Blanco (hybrid); ‘Hamlin’ orange, C. sinensis (L.) Osbeck; and pummelo, C. grandis (L.) Osbeck. Following eclosion, 100% of adults developed in 7.6 to 7.9 days when pollen of ice plant, live oak, castor bean or cattail was provided as single food sources at 27 ± 1°C. A total of 30.8 and 30.4 eggs per female were obtained when ice plant and live oak pollens, respectively, were provided compared to 23.0 and 19.2 eggs per female when castor bean and cattail pollens were used. Life table parameters were: mean generation time (T) = 12.38; net reproductive rate (Ro) = 17.17; intrinsic rate of natural increase (rm) = 0.2296 and finite rate of increase (λ) = 1.258 when live oak pollen was used as the food source compared with T = 12.47, Ro = 17.79, rm = 0.2308 and λ = 1.260 for ice plant pollen. Castor bean and cattail pollens are available as potential food sources for E. mesembrinus, but they are not as effective as ice plant or live oak pollens. Larvae of E. mesembrinus required 8.5 days to develop to adults, while only 72% of the larvae survived when B. pilosa pollen was used as the food source. Each female deposited an average of 0.45 eggs per day during the first 5 days of oviposition. Citrus pollens including C. paradisi, C. reticulata, C. sinensis and C. grandis are available as food sources for E. mesembrinus, and survivorship ranged from 64 to 84%. Developmental time was prolonged (8.8 to 12.5 days) and fecundity was also very low (0.10 to 0.67 eggs/female/day) during the first 5 days of oviposition compared with both ice plant and live oak pollens.
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Methyl anthranilate (MA) contents of 159 samples of Florida honey from 10 crop years are summarized (mean, 2.79; range, 0−5.04 ppm). A highly significant relationship between MA and citrus pollen content is shown for 85 samples from 2 crop years. Sixty-three samples of “monofloral” Florida citrus honeys from two crops averaged 64% citrus pollen and 3.1 ppm of MA. Keywords: Citrus honey; methyl anthranilate; pollen analysis
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Caffeine was isolated and identified in extracts from flower buds of several citrus cultivars and from leaves of Valencia oranges (Citrus sinensis L. Osbeck). No caffeine was detected in orange juice. Identification was by high-performance liquid chromatography (HPLC), gas chromatography (GC), ultraviolet spectroscopy (UV), and mass spectra (MS).